Department of Physics, University of MarylandCollege Park, MD, United States.
Department of Biology, University of MarylandCollege Park, MD, United States.
Front Neural Circuits. 2017 Aug 15;11:56. doi: 10.3389/fncir.2017.00056. eCollection 2017.
The application of 2-photon laser scanning microscopy (TPLSM) techniques to measure the dynamics of cellular calcium signals in populations of neurons is an extremely powerful technique for characterizing neural activity within the central nervous system. The use of TPLSM on awake and behaving subjects promises new insights into how neural circuit elements cooperatively interact to form sensory perceptions and generate behavior. A major challenge in imaging such preparations is unavoidable animal and tissue movement, which leads to shifts in the imaging location (jitter). The presence of image motion can lead to artifacts, especially since quantification of TPLSM images involves analysis of fluctuations in fluorescence intensities for each neuron, determined from small regions of interest (ROIs). Here, we validate a new motion correction approach to compensate for motion of TPLSM images in the superficial layers of auditory cortex of awake mice. We use a nominally uniform fluorescent signal as a secondary signal to complement the dynamic signals from genetically encoded calcium indicators. We tested motion correction for single plane time lapse imaging as well as multiplane (i.e., volume) time lapse imaging of cortical tissue. Our procedure of motion correction relies on locating the brightest neurons and tracking their positions over time using established techniques of particle finding and tracking. We show that our tracking based approach provides subpixel resolution without compromising speed. Unlike most established methods, our algorithm also captures deformations of the field of view and thus can compensate e.g., for rotations. Object tracking based motion correction thus offers an alternative approach for motion correction, one that is well suited for real time spike inference analysis and feedback control, and for correcting for tissue distortions.
双光子激光扫描显微镜（TPLSM）技术在神经元群体中测量细胞钙信号动力学的应用是一种非常强大的技术，可用于描述中枢神经系统内的神经活动。在清醒和行为的动物上使用 TPLSM 有望为理解神经回路元件如何协同相互作用以形成感觉知觉和产生行为提供新的见解。在对这些标本进行成像时，一个主要的挑战是不可避免的动物和组织运动，这会导致成像位置的偏移（抖动）。图像运动的存在会导致伪影，特别是因为 TPLSM 图像的定量分析涉及到对每个神经元的荧光强度波动的分析，这些波动是从小的感兴趣区域（ROI）中确定的。在这里，我们验证了一种新的运动校正方法，以补偿清醒小鼠听觉皮层浅层 TPLSM 图像的运动。我们使用名义上均匀的荧光信号作为补充遗传编码钙指示剂的动态信号的辅助信号。我们测试了用于单平面延时成像以及皮质组织的多平面（即体积）延时成像的运动校正。我们的运动校正程序依赖于找到最亮的神经元，并使用已建立的粒子发现和跟踪技术随时间跟踪它们的位置。我们表明，我们的基于跟踪的方法提供了亚像素分辨率，而不会牺牲速度。与大多数已建立的方法不同，我们的算法还捕获视场的变形，因此可以补偿例如旋转。基于对象跟踪的运动校正因此提供了一种替代的运动校正方法，非常适合实时尖峰推断分析和反馈控制，以及校正组织变形。
